Cytotoxicity is a key strategy utilized by natural killer (NK) and T cells to kill tumor cells, but its molecular basis is unclear. Analysis of signal molecules indicated that mitogen-activated protein kinases (MAPK)/ERK2 critically controlled direct NK lysis. Tumor ligation in human NK cells induced MAPK tyrosine phosphorylation and enzymatic activity. Moreover, MAPK inactivation by pharmacological inhibition with PD098059 or dominant-negative ERK2 expression interfered with NK lysis of 51-Cr labeled tumor cells, and interrupted perforin/granzyme B polarization. It is thus important to define what signal molecules control MAPK activation, with the goal of possibly finding means to enhance this pathway in cancer patients, once it is identified. Preliminary studies indicated that NK utilized a ras-independent MAPK cascade to lyse tumor cells. Farnesyl transferase inhibitor, FTI-277, or dominant-negative N17ras expression, had no deleterious effect on NK lysis of tumor cells, perforin/granzyme B mobilization, or MAPK activation. Instead, tumor cells activate a ras related protein, rap1A, to its GTP form, in NK cells. GGTI-298, which blocks geranylation, a process used by rap1A for membrane localization, effectively inhibited NK lytic capacity and rap1A activation. This proposal is to test the hypothesis that rap1A is an upstream effector of MAPK, and Aim 1 is to identify if the rap1A/B-raf/MEK/MAPK pathway critically controls the lytic process. Alternatively, other pathways may feed into MAPK to represent overlapping or separate functions. Aim 2 is analyze the roles of PI 3-kinase and Syk70 in MAPK activation and Aim 3 addresses the hypothesis that inhibitory NK receptors, such as KIR, inactivate function via MAPK. Aim 4 is to identify if MAPK control of perforin movement is via regulation of microtubule reorganization. The experimental plan follows an in-depth biochemical and genetic approach, using pharmacological agents and mutant gene constructs, to identify specific plays in MAPK activation. Identification of a distinct ras- independent NK lytic process could provide a new strategy for therapeutics that specifically targets ras-transformed tumors and not NK function in patients.